This invention relates generally to apparatus and methods for use in conducting chemical, including biochemical, reactions on a solid substrate. More particularly, the invention relates to apparatus and methods for conducting various processing steps that are part of such chemical reactions. The invention has utility in fields relating to biology, chemistry and biochemistry. The invention has particular application to the area of analyzing the results of hybridization reactions involving nucleic acids and proteins.
Determining the nucleotide sequences and expression levels of nucleic acids (DNA and RNA) is critical to understanding the function and control of genes and their relationship, for example, to disease discovery and disease management. Analysis of genetic information plays a crucial role in biological experimentation. This has become especially true with regard to studies directed at understanding the fundamental genetic and environmental factors associated with disease and the effects of potential therapeutic agents on the cell. Such a determination permits the early detection of infectious organisms such as bacteria, viruses, etc.; genetic diseases such as sickle cell anemia; and various cancers. New methods of diagnosis of diseases, such as AIDS, cancer, sickle cell anemia, cystic fibrosis, diabetes, muscular dystrophy, and the like, rely on the detection of mutations present in certain nucleotide sequences. This paradigm shift has lead to an increasing need within the life science industries for more sensitive, more accurate and higher-throughput technologies for performing analysis on genetic material obtained from a variety of biological sources.
Unique or misexpressed nucleotide sequences in a polynucleotide can be detected by hybridization with a nucleotide multimer, or oligonucleotide, probe. Hybridization reactions between surface-bound probes and target molecules in solution may be used to detect the presence of particular biopolymers. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. These techniques rely upon the inherent ability of nucleic acids to form duplexes via hydrogen bonding according to Watson-Crick base-pairing rules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen-bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research. An oligonucleotide probe employed in the detection is selected with a nucleotide sequence complementary, usually exactly complementary, to the nucleotide sequence in the target nucleic acid. Following hybridization of the probe with the target nucleic acid, any oligonucleotide probe/nucleic acid hybrids that have formed are typically separated from unhybridized probe. The amount of oligonucleotide probe in either of the two separated media is then tested to provide a qualitative or quantitative measurement of the amount of target nucleic acid originally present.
Such reactions form the basis for many of the methods and devices used in the new field of genomics to probe nucleic acid sequences for novel genes, gene fragments, gene variants and mutations. The ability to clone and synthesize nucleotide sequences has led to the development of a number of techniques for disease diagnosis and genetic analysis. Genetic analysis, including correlation of genotypes and phenotypes, contributes to the information necessary for elucidating metabolic pathways, for understanding biological functions, and for revealing changes in genes that confer disease. Many of these techniques generally involve hybridization between a target nucleotide sequence and a complementary probe, offering a convenient and reliable means for the isolation, identification, and analysis of nucleotides. The surface-bound probes may be oligonucleotides, peptides, polypeptides, proteins, antibodies or other molecules capable of reacting with target molecules in solution.
Direct detection of labeled target nucleic acid hybridized to surface-bound polynucleotide probes is particularly advantageous if the surface contains a mosaic of different probes that are individually localized to discrete, known areas of the surface. Such ordered arrays of probes are commonly referred to as “biochip” arrays. Biochip arrays containing a large number of oligonucleotide probes have been developed as tools for high throughput analyses of genotype and gene expression. Oligonucleotides synthesized on a solid support recognize uniquely complementary nucleic acids by hybridization, and arrays can be designed to define specific target sequences, analyze gene expression patterns or identify specific allelic variations.
In one approach, cell matter is lysed, to release its DNA as fragments, which are then separated out by electrophoresis or other means, and then tagged with a fluorescent or other label. The resulting DNA mix is exposed to an array of oligonucleotide probes, whereupon selective attachment to matching probe sites takes place. The array is then washed and imaged so as to reveal for analysis and interpretation the sites where attachment occurred.
One typical method involves hybridization with probe nucleotide sequences immobilized in an array on a substrate having a surface area of typically less than a few square centimeters. The substrate may be glass, fused silica, silicon, plastic or other material; preferably, it is a glass slide, which has been treated to facilitate attachment of the probes. The mobile phase, containing reactants that react with the attached probes, is placed in contact with the substrate, covered with another slide, and placed in an environmentally controlled chamber such as an incubator. Normally, the reactant targets in the mobile phase diffuse through the liquid to the interface where the complementary probes are immobilized, and a reaction, such as a hybridization reaction, then occurs. Preferably, the mobile phase targets are labeled with a detectable tag, such as a fluorescent tag, or chemiluminescent tag, or radioactive label, so that the reaction can be detected. The location of the signal in the array provides the target identification. The hybridization reaction typically takes place over a time period of seconds up to many hours.
Biochip arrays have become an increasingly important tool in the biotechnology industry and related fields. These binding agent arrays, in which a plurality of binding agents are synthesized on or deposited onto a substrate in the form of an array or pattern, find use in a variety of applications, including gene expression analysis, drug screening, nucleic acid sequencing, mutation analysis, and the like. Substrate-bound biopolymer arrays, particularly oligonucleotide, DNA and RNA arrays, may be used in screening studies for determination of binding affinity and in diagnostic applications, e.g., to detect the presence of a nucleic acid containing a specific, known oligonucleotide sequence.
Polynucleotide microarrays may be subjected to hybridization reactions in the following manner. In one approach polynucleotide target material is prepared by purifying, labeling and suspending it in liquid hybridization buffer, which is typically a solution of lithium lauryl sulfate, LiCl, Li-MES, EDTA and Triton X-100 in various percentage concentrations. This hybridization buffer mix is applied to the active surface of the microarray and the microarray is incubated for a period of time, typically about 18 hours at an elevated temperature of about 40 to about 70° C. Following incubation, the hybridization buffer is removed from the microarray surface and the non-specifically bound target material is washed away in one or more wet process steps using one or more wash reagents of varying stringency. Stringency is controlled typically through salt concentration and reagent temperature. Following the last wash step, the surfaces of the microarray are dried. The microarray is fluorescently scanned and the resulting image is analyzed to determine the degree of probe-target binding.
The pattern of binding by target molecules to biopolymer probe spots on the microarray or biochip forms a pattern on the surface of the biochip and provides desired information about the sample. Hybridization patterns on biochip arrays are typically read by optical means, although other methods may also be used. For example, laser light in the Agilent Technologies Inc. Microarray Scanner excites fluorescent molecules incorporated into the nucleic acid probes on a biochip, generating a signal only in those spots on the biochip that have a target molecule bound to a probe molecule, thus generating an optical hybridization pattern. This pattern may be digitally scanned for computer analysis. Such patterns can be used to generate data for biological assays such as the identification of drug targets, single-nucleotide polymorphism mapping, monitoring samples from patients to track their response to treatment, and assess the efficacy of new treatments.
The process steps associated with hybridizing microarrays (incubation, washing and drying) are commonly performed manually using conventional molecular biology/chemical laboratory apparatus and techniques. The above techniques are particularly applicable for microscope-slide-like microarrays. Incubation is done statically or with vibrating motion in heated water baths or with gentle rotational mixing motion in dry incubators. Washing is done using histology slide staining glassware, open dishes and tubs, and hot plates with magnetic stirrers to agitate the wash buffer. Drying is done by applying a stream of compressed dry gas (air or nitrogen) to the surface of the microarray or by centrifuging the microarray.
Significant process variability is inherent in the manual hybridization of microarrays, especially manual washing and drying. This leads to poor reproducibility. Manual methods result in overall degraded microarray performance as evidenced by variable sensitivity, poor spatial uniformity, higher background levels, poor background level spatial uniformity, reduced expressed-signal-to-background ratio, and variable specificity. Specifically, manual methods are burdened by the following characteristics: a) multiple repetitive process steps which are labor intensive, b) significant touching and handling of the individual microarray support substrates as they move from one process step to the next, c) exposure of the microarray support substrates to the surrounding laboratory atmosphere as they are transferred from one process step to the next, d) inability to keep substrates completely wet (submerged) at critical times during processing, e) exposure of the microarray support substrates to wash reagents of indeterminate stringency and concentration due to poorly controlled wash bath temperatures (wash baths are commonly heated and stirred on laboratory hot plates without the benefits of closed-loop temperature control), f) loosely controlled wash step duration and variability of wash timing due to the difficulty of manually managing a time-staggered queue of microarray support substrates as they are processed, g) gradual degradation of wash reagents due to contaminant build-up caused by processing multiple support substrates with a single replenishment of reagent, which is aggravated by the inconvenience of manually replacing pre-heated or pre-cooled reagents.
The process steps associated with hybridizing microarrays (incubation, washing and drying) have been automated to varying degrees by laboratory instrumentation designers. There are numerous examples of commercially available hybridization stations. In general, the purpose of every commercial automated hybridization station, whether for packaged microarrays or for microscope-slide-like microarrays, is to provide consistent, regimented incubation, washing and drying. The Fluidics Station from Affymetrix, Inc., provides automated hybridization for their GeneChip® series of packaged microarrays. The GeneChip® series of microarrays represent a ‘closed system’ since they are mechanically incompatible with conventional microscope-slide-like microarrays. Some currently available hybridization stations capable of processing microscope-slide-like microarrays are: the Automatic Slide Processor (ASP) from Amersham Pharmacia BioScience; the GeneTAC™ HybStation and Hyb4 hyb-station, both from Genomics Solutions Inc.; the TECAN HS-Series HybStation from TECAN (Austria); the a-Hyb™ from Memorec Stoffel GmbH; the DISCOVERY™ system from Ventana; and the OmniSlide Modular System from Hybaid UK (a Themmo BioAnalysis Company).
Most of the commercially available hybridization stations have one or more limitations such as: poor drying of microarrays by controlled fluid draining, by dry gas injection or by hybridization chamber evacuation; inability to handle multiple isolated arrays per substrate; poor accommodation of wide range of slide dimensions; lack of visual inspection during sample loading; no provision for pre-heating or pre-cooling reagent reservoirs, poor or no reagent cooling capability, reduced wash effectiveness due to moderate to low volume washing; poor dilution of non-specifically bound target material and spent wash buffer residue due to low fluid volumes.
In addition, hybridization stations have been disclosed in U.S. patent application Ser. No. 09/919,073, filed Jul. 30, 2001 (Donlon, et al.), entitled “Sample Processing Apparatus and Methods,” and in U.S. Patent Application 2000/004,6702 A1, filed 19 Jun. 2001 (Schembri), entitled “Devices for Performing Array Hybridization Assays and Methods of Using the Same.”
In addition, hybridization designs are disclosed in patent application WO 01/32934 A2, Hybridization Station, assigned to Arcturus Engineering Inc, and patent application WO 01/31347 A1, Modular Automated Sample Processing Apparatus, assigned to SmithKline Beecham PLC.
There is a need for an apparatus and methods for conducting processing steps in chemical reactions, particularly on a non-porous substrate, which may avoid or alleviate one or more of the aforementioned limitations.